1. Field of the Invention
This invention relates generally to novel mammalian apoptosis-inducing factor (AIF) polypeptides, DNAs encoding for the AIF polypeptides, and uses thereof.
2. Related Technology
Apoptosis is essential for the maintenance of tissue size and cell number homeostasis of multi-cellular organisms, and apoptotic abnormalities are thought to play an important role in the development of various neoplastic diseases as well as a number of neurodegenerative diseases.
Mitochondria play a key role in the regulation of apoptosis. A variety of key events in apoptosis involve mitochondria, including the release of caspase activators (such as cytochrome c), changes in electron transport, loss of mitochondrial transmembrane potential (thus allowing several proteins found within the mitochondrial intermembrane space to be liberated through the outer mitochondrial membrane, thereby participating in the apoptotic degradation phase), altered cellular oxidation-reduction, and which involves the of pro- and anti-apoptotic Bcl-2 family of proteins. The different signals that converge on mitochondria to trigger or inhibit these events and their downstream effects delineate several major pathways in physiological cell death.
Because apoptosis and its regulation plays a critical role in the maintenance of cellular and tissue homeostasis, there exists a need to develop material and methods for either inducing or inhibiting apoptosis as well as to provide systems for screening for additional candidate substances that either induce or inhibit apoptosis.
The present invention is directed to isolated polynucleotides encoding a mammalian apoptosis-inducing factor or biologically active conserved variants, allelic variants, isoforms, analogs, and fragments thereof, wherein the polynucleotides are selected from the group consisting of cDNA, genomic DNA, and chemically synthesized DNA. In another aspect the invention is directed isolated polynucleotides encoding murine apoptosis-inducing factor or biologically active conserved variants, allelic variants, isoforms, analogs, and fragments thereof. The invention is also directed to isolated polynucleotides encoding human apoptosis-inducing factor or biologically active conserved variants, allelic variants, isoforms, analogs, and fragments thereof. As yet another aspect, the present invention is directed to isolated polynucleotides that encode a polypeptide having at least 70-95 percent identity to the polypeptides comprising the amino acid sequences set forth in SEQ ID NOS: 2, 3, 5, 6, 8, 9, 11, 12, 14, and 15.
The present invention is also directed to isolated polynucleotides encoding a mammalian apoptosis-inducing factor the polynucleotide being selected from the group consisting of (a) the DNA molecules set forth in SEQ ID NOS: 1, 4, 7, 10, 13, DNA molecules encoding variants including conserved variants, allelic variants, analogs, and fragments thereof; (b) DNA molecules which hybridize, under high stringency conditions, to the DNA molecules defined in (a) or hybridizable fragments thereof; and (c) DNA molecules that code an expression for the amino acids encoded by any of the foregoing DNA molecules.
In another of its aspects, the present invention is directed to expression vectors or cloning vectors comprising any of the disclosed AIF-encoding polynucleotides as well to host cells transformed with any of the disclosed AIF-encoding polynucleotides.
The present invention is also directed to mammalian cells containing a mammalian apoptosis-inducing factor encoding DNA modified so as to permit higher expression of the apoptosis-inducing factor by means of a homologous recombinational event consisting of inserting an expression regulatory sequence in functional proximity to the apoptosis-inducing factor encoding DNA, wherein the inserted expression regulatory sequence is not a native apoptosis-inducing factor expression regulatory sequence.
As yet another aspect, the present invention is directed to methods for producing an apoptosis-inducing factor polypeptide, the method comprising the steps of: (a) culturing a host cell according to claim 20 or 23 under conditions suitable for the expression of the apoptosis-inducing factor polypeptide; and (b) recovering the expressed apoptosis-inducing factor polypeptide.
The present invention is also directed an isolated purified mammalian apoptosis-inducing factor and biologically active conserved variants, allelic variants, isoforms, analogs, and fragments thereof. The present invention is also directed to an isolated purified murine apoptosis-inducing factor and biologically active conserved variants, allelic variants, isoforms, analogs, and fragments thereof. The present invention is further directed to an isolated purified human apoptosis-inducing factor and biologically active conserved variants, allelic variants, isoforms, analogs, and fragments thereof.
As yet a further aspect of the present invention is directed antibody substances which specifically bind the disclosed apoptosis-inducing factors.
The present invention is also directed to derivatives of the disclosed apoptosis-inducing factors.
The present invention is also directed to methods for determining the presence of mammalian apoptosis-inducing factor in a biological sample comprising the steps of: (a) obtaining a biological sample; (b) exposing said biological sample to a mammalian apoptosis-inducing factor-specific antibody; and (c) detecting the binding of mammalian apoptosis-inducing factor-specific antibody in said biological sample. The present invention is also directed to methods for determining the presence of mammalian apoptosis-inducing factor-specific polynucleotide molecules in a biological sample comprising the steps of: (a) collecting a biological sample;(b) isolating polynucleotide molecules from said biological sample; (c) hybridizing to said polynucleotide molecules a diagnostic reagent; and (d) detecting the binding of the mammalian apoptosis-inducing factor-specific polynucleotide molecules in said biological samples.
The present invention is also directed to methods for determining the presence of mammalian-apoptosis inducing factor-specific polynucleotide molecule in a tissue or cellular sample comprising the steps of: (a) collecting tissue or cellular sample;(b) hybridizing said tissue or cellular sample to a diagnostic reagent; and (c) detecting the binding of the mammalian apoptosis-inducing factor-specific polynucleotide molecules in the tissue or cellular sample to said diagnostic reagent.
As yet another aspect of the invention is directed to methods of identifying a candidate inhibitor of mammalian apoptosis-inducing factor binding to a mammalian apoptosis-inducing factor binding protein comprising the steps of: (a) exposing mammalian apoptosis-inducing factor to a mammalian apoptosis-inducing factor binding protein under conditions which permit binding of mammalian apoptosis-inducing factor to a mammalian apoptosis-inducing factor binding protein in the presence or absence of a candidate inhibitor; (b) measuring the binding of mammalian apoptosis-inducing factor to a mammalian apoptosis-inducing factor binding protein in the presence or absence of the candidate inhibitor; (c) comparing the level of binding observed in step (a); and (d) identifying the compound as an inhibitor of mammalian apoptosis-inducing factor binding by its ability to prevent binding of mammalian apoptosis-inducing factor to a mammalian apoptosis-inducing factor binding protein.
The present invention is also directed to a composition comprising an isolated purified mammalian apoptosis-inducing factor or biologically active conserved variants, allelic variants, isoforms, analogs, and fragments thereof and an acceptable carrier, diluent and/or adjuvant. The present invention is also directed to a murine apoptosis-inducing factor or biologically active conserved variants, allelic variants, isoforms, analogs, and fragments thereof and an acceptable carrier, diluent and/or adjuvant. The present invention is also directed to a human apoptosis-inducing factor or biologically active conserved variants, allelic variants, isoforms, analogs, and fragments thereof and an acceptable carrier, diluent and/or adjuvant.
As yet another aspect of the invention is directed to methods of inhibiting cell proliferation via administration of any of the disclosed apoptosis-inducing factors or compositions thereof.
Other objectives and advantages of the invention may be apparent to those skilled in the art from a review of the following detailed description, including any drawings, as well as the approved claims.